Seeinstructions): The failure of an HIVT-cell vaccine in two recent human trials raises two questions that can be addressed by this project. 1) Is a replication-impaired adenovirus vectored-vaccine expressing HIV gag, pol and nef, and inoculated systemically in 3 doses, immunogenic at mucosal surfaces? In other words, do vaccine-specific T cells, elicited by systemic immunization with a non-replicating vector, distribute throughout the body sufficiently to protect a mucosal surface? No currently used vaccine has been shown to work in this way and in this sense, the STEP trial was a novel and innovative experiment. 2) Does pre-existing adenovirus immunity significantly decrease the strength or quality of the HIV-specific T cell response in mucosal tissues? If so, perhaps pre-existing immunity to adenovirus reduced the HIV-specific T cell response in mucosal tissues below a critical threshold. However, this would not account for a possible increased susceptibility to HIV infection in vaccinees with pre-existing adenovirus immunity. To address this question experimentally, both Projects #1 and 2 are required. Using a well-defined SIV-macaque animal model, the animal experiments designed for this program- project, both the adenoviral vaccine modelling experiments and the mechanism studies, will allow these questions of vaccine-induced cellular immunity to be addressed directly, which could not be done using human subjects. Our specific hyopthesis for this project on the T cell vaccine-induced immune response is that a replication-impaired, adenovirus-vectored SIV gag/pol/nef vaccine inoculated by the intramuscular route will induce strong CD8+ T cell responses detected in the blood but weaker and less functional responses in genital and rectal mucosa. Further, pre-existing adenovirus immunity will reduce the T cell responses in mucosal sites below a critical threshold for protection. There are two specific aims: 1) to determine the relative strength and quality of SIV-specific T cell immunity elicited by vaccination in blood, mucosal tissues and secondary lymphoid organs, and 2) to compare the strength and quality of SIV-specific T cell responses in each tissue of vaccinated animals with or without pre-existing adenoviral immunity. Thus, this project addresses the relative immunogenicity of a specific, nonreplicating vector vaccine in the body of the host, in the presence or absence or vector immunity that is a barrier to immunization. These are compelling issues that will need to be addressed by any future candidate HIV vaccine. RELEVANCE (See instructions): There are formidable obstacles to developing an HIV vaccine, including the high-level replication of this persistent virus, the high rate of mutation of the viral genome and the immune dysfunction that the infection inevitably causes. The SIV/macaque animal model of HIV infection and AIDS, while not a perfect model, is needed to address critical, basic questions of vaccine efficacy and safety before human vaccine trials.